Protein kinase D (PKD) has recently emerged as a family of enzymes with important cardiac actions. Most studies have focused on PKD1 (the founding member of this enzyme family) which phosphorylates HDAC5, a class II histone deacetylase that regulates chromatin remodeling and hypertrophy. PKD1 also phosphorylates CREB and sarcomeric proteins (such as cardiac troponin I and cardiac myosin-binding protein C), but their role in cardiac growth responses has not adequately been considered. Similarly, many PKD1 substrates also are phosphorylated by PKD2 or PKD3, but the notion that PKD2 or PKD3 cooperate with PKD1 to regulate cardiac remodeling also has not been considered. Our preliminary studies showing that PKD1, 2, and 3 have similar (but not identical) in vitro substrate specificities, PKD1 and PKD2 are activated in a stimulus-specific manner in cardiomyocytes, and PKDs have both overlapping and non-redundant roles in mouse embryonic development and adult heart hypertrophy provide the rationale for the aims of this application. Aim #1 will use state-of-the- art peptide library screens to define PKD isoform substrate specificity. This aim also will use biochemical approaches and D Kinase Activity Reporter assays to identify activation mechanisms and signaling repertoires of individual PKD isoforms. We will use gene knockout and overexpression strategies (including with analog- sensitive PKD mutants) to identify PKD isoform-specific substrates and effectors in cardiomyocytes. Aim #2 will use PKD1, 2, and 3 single global knockout mice and PKD1/D2, PKD1/D3, and PKD2/D3 global and cell type specific double knockout mice to analyze the role of PKDs in cardiovascular development. Inducible adult cardiac specific PKD knockout mice and a transgenic analogue-sensitive PKD1 mutant mouse also will be generated to examine the role of PKDs in the adult myocardium. The overarching goal of these studies is to identify novel regulatory controls and cardiac actions of PKDs that can be targeted to prevent or slow the evolution of cardiac hypertrophy and heart failure phenotypes.